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Preparation of Uniformly Oriented Inverted Inner (Cytoplasmic) Membrane Vesicles from Gram-Negative Bacterial Cells

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Bacterial Secretion Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2715))

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Abstract

The complex double-membrane organization of the envelope in Gram-negative bacteria places unique biosynthetic and topological constraints that can affect translocation of lipids and proteins synthesized on cytoplasm facing leaflet of cytoplasmic (inner) membrane (IM), across IM and between IM and outer membrane (OM). Uniformly oriented inside-out (ISO) vesicles became functional requisite for many biochemical reconstitution functional assays, vectorial proteomics, and vectorial lipidomics. Due to these demands, it is necessary to develop simple and reliable approaches for preparation of uniformly oriented IM membrane vesicles and validation of their sidedness. The uniformly ISO oriented membrane vesicles which have the cytoplasmic face of the membrane on the outside and the periplasmic side facing the sealed lumen can be obtained following intact cell disruption by a single passage through a French pressure cell (French press) at desired total pressure. Although high-pressure lysis leads to the formation of mostly inverted membrane vesicles (designated and abbreviated usually as ISO vesicles, everted or inverted membrane vesicles (IMVs)), inconclusive results are quite common. This uncertainty is due mainly by applying a different pressures, using either intact cells or spheroplasts and presence or absence of sucrose during rupture procedure. Many E. coli envelope fractionation techniques result in heterogeneity among isolated IM membrane vesicles. In part, this is due to difficulties in simple validation of sidedness of oriented membrane preparations of unknown sidedness. The sidedness of various preparations of membrane vesicles can be inferred from the orientation of residing uniformly oriented transmembrane protein. We outline the method in which the orientation of membrane vesicles can be verified by mapping of uniform or mixed topologies of essential protein E. coli protein leader peptidase (LepB) by advanced SCAM™. Although the protocol discussed in this chapter has been developed using Escherichia coli and Yersinia pseudotuberculosis, it can be directly adapted to other Gram-negative bacteria including pathogens.

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Acknowledgments

This work was supported by NIH grant R01GM121493-6, European Union Marie Skłodowska-Curie Grant H2020-MSCA-RISE-2015-690853, and NATO Science for Peace and Security Programme-SPS 98529.

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  1. Department of Biochemistry & Molecular Biology, University of Texas Health Science Center at Houston, McGovern Medical School, Houston, TX, USA

    Mikhail Bogdanov

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  1. Mikhail Bogdanov
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Correspondence to Mikhail Bogdanov .

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  1. Laboratoire d’Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ, CNRS, Marseille, France

    Laure Journet

  2. Laboratoire d’Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ, CNRS, Marseille, France

    Eric Cascales

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Bogdanov, M. (2024). Preparation of Uniformly Oriented Inverted Inner (Cytoplasmic) Membrane Vesicles from Gram-Negative Bacterial Cells. In: Journet, L., Cascales, E. (eds) Bacterial Secretion Systems . Methods in Molecular Biology, vol 2715. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3445-5_10

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